Sleeping beauty, a transposon vector with a broad host range for the genetic transformation in vertebrates

ABSTRACT

The invention relates to the use of the gene transfer system Sleeping Beauty for the somatic gene transfer for the purpose of stably inserting DNA in the chromosomes of living vertebrates, comprising the two components of the transfer system Sleeping Beauty that are injected into the somatic cells of an animal for the purpose of gene therapy.

Considerable effort has been devoted to the development of in vivo genedelivery strategies for the treatment of inherited and aquired disordersin humans (somatic gene transfer) as well as for transgenesis of certainvertebrate species for agricultural and medical biotechnology (germlinegene transfer). For effective gene therapy it is necessary to: 1)achieve delivery of therapeutic genes at high efficiency specifically torelevant cells, 2) express the gene for a prolonged period of time, 3)ensure that the introduction of the therapeutic gene is not deleterious.

There are several methods and vectors in use for gene delivery for thepurpose of human gene therapy (Verma and Somia,1997). These methods canbe broadly classified as viral and nonviral technologies, and all haveadvantages and limitations; none of them providing a perfect solution.In general, vectors that are able to integrate the transgene have thecapacity to provide prolonged expression as well. On the other side,random integration into chromosomes is a concern, because of thepotential disruption of endogenous gene function at and near theinsertion site.

Adapting viruses for gene transfer is a popular approach, but geneticdesign of the vector is restricted due to the constraints of the virusin terms of size, structure and regulation of expression. Retroviralvectors (Miller, 1997) are efficient at integrating foreign DNA into thechromosomes of transduced cells, and have enormous potential forlife-long gene expression. However, the amount of time and financialresources required for their preparation may not be amenable toindustrial-scale manufacture. Furthermore, there are several otherconsiderations including safety, random chromosomal integration and therequirement of cell replication for integration. Lentiviral systems,based on the human immunodeficiency virus (HIV) belong to retroviruses,but they can infect both dividing and non-dividing cells. Adenovirusvectors have been shown to be capable of in vivo gene delivery oftransgenes to a wide variety of both dividing and non-dividing cells, aswell as mediating high level, but short term transgene expression.Adenoviruses lack the ability to integrate the transferred gene intochromosomal DNA, and their presence in cells is short-lived. Thus,recombinant adenovirus vectors have to be administered repeatedly,generating an undesirable immune response in humans, due to theimmunogenity of the vector. Adeno Associated Virus (AAV) vectors haveseveral potential advantages to be explored, including the potential oftargeted integration of the transgene. One of the obvious limitations ofthe AAV vehicle is the low maximal insert size (3.5-4.0 kb). Currently,combination (hybrid) vectors (retroviral/adenoviral, retroviral/AAV,etc.) have been developed that are able to address certain problems ofthe individual viral vector systems.

Nonviral methods, including DNA condensing agents, liposomes,microinjection and “gene guns” might be easier and safer to use thanviruses. However, the efficiency of naked DNA entry and uptake is low,that can be increased by using liposomes. In general, the currently usednon-viral systems are not equipped to promote integration intochromosomes. As a result, stable gene transfer frequencies usingnonviral systems have been very low. Moreover, most nonviral methodsoften result in concatamerization as well as random breaks in input DNA,which might lead to gene silencing.

PROBLEM TO BE ADDRESSED

Currently, there is no gene delivery system in vertebrates for somaticand germline gene transfer which would combine the followingcharacteristics: 1) ability to transfer genes in vivo; 2) wide host- andtissue-range; 3) stable insertion of genes into chromosomes; 3)faithful, long-term expression of transferred genes; 4) safety; 5)cost-effective large-scale manufacture.

DESCRIPTION

Transposable elements, or transposons in short, are mobile segments ofDNA that can move from one locus to another within genomes (Plasterk etal., 1999). These elements move via a conservative, “cut-and-paste”mechanism: the transposase catalyzes the excision of the transposon fromits original location and promotes its reintegration elsewhere in thegenome. Transposase-deficient elements can be mobilized if thetransposase is provided in trans by another transposase gene. Thus,transposons can be harnessed as vehicles for bringing new phenotypesinto genomes by transgenesis. They are not infectious and due to thenecessity of adaptation to their host, they thought to be less harmfulto the host than viruses.

DNA transposons are routinely used for insertional mutagenesis, genemapping, and gene transfer in well-established, non-vertebrate modelsystems such as Drosophila melanogaster or Caenorhabditis elegans, andin plants. However, transposable elements have not been used for theinvestigation of vertebrate genomes for two reasons. First, until now,there have not been any well-defined, DNA-based mobile elements in thesespecies. Second, in animals, a major obstacle to the transfer of anactive transposon system from one species to another has been that ofspecies-specificity of transposition due to the requirement for factorsproduced by the natural host.

Sleeping Beauty (SB) is an active Tc1-like transposon that wasreconstructed from bits and pieces of inactive elements found in thegenomes of teleost fish. (SB) is currently the only active DNA-basedtransposon system of vertebrate origin that can be manipulated in thelaboratory using standard molecular biology techniques. SB mediatesefficient and precise cut-and-paste transposition in fish, frog, andmany mammalian species including mouse and human cells (Ivics et al.,1997; Luo et al., 1998; Izsvak et al., 2000; Yant et al., 2000).

Some of the main characteristics of a desirable transposon vector are:ease of use, relatively wide host range, little size or sequencelimitations, efficient chromosomal integration, and stable maintenanceof faithful transgene expression throughout multiple generations oftransgenic cells and organisms. Sleeping Beauty fulfills theserequirements based on the following findings.

Experimental Results

Sleeping Beauty is active in diverse vertebrate species. To assess thelimitations of host specificity of SB among vertebrates, cultured cellsof representatives of different vertebrate classes were subjected to ourstandard transposition assay. Cell lines from seven different fishspecies, three from mouse, two from human and one each from a frog, aquail, a sheep, a cow, a dog, a rabbit, a hamster and a monkey weretested. As summarized in Table 1, SB was able to increase the frequencyof transgene integration in all of these cell lines, with the exceptionof the quail. Thus, we concluded that SB would be active in essentiallyany vertebrate species (Izsvak et al., 2000).

Effects of transposon size on the efficiency of Sleeping Beautytransposition. The natural size of SB is about 1.6 kb. To be useful as avector for somatic and germline transformation, a transposon vector mustbe able to incorporate large (several kb) DNA fragments containingcomplete genes, and still retain the ability to be efficiently mobilizedby a transposase. In order to determine the size-limitations of the SBsystem, a series of donor constructs containing transposons ofincreasing length (2.2; 2.5; 3.0; 4.0; 5.8; 7.3 and 10.3 kb) was tested.Similarly to other transposon systems, larger elements transposed lessefficiently, and with each kb increase in transposon length we found anexponential decrease of approximately 30% in efficiency of transposition(FIG. 1) (Izsvak et al., 2000). The maximum size of SB vectors,similarly to most retroviral vectors, was found to be about 10 kb.However, although efficiency of transposition appears to decrease withincreasing vector size as a general rule, the upper limit does notappear to be as strict as for retroviral vectors. Moreover, a decreaseof length of DNA outside the transposon increases the efficiency oftransposition as a general rule (˜30% increase/kb) (Izsvak et al.,2000). In other words, at a given insert size the transpositionefficiency can be increased by bringing the two inverted repeats of thetransposon closer on a circular plasmid molecule.

A 14 kb piece of DNA, flanked by a pair of Paris elements, appears tohave transposed in Drosophila virilis. We hypothesized that this kind of“sandwich” arrangements of two complete SB elements flanking a transgenewill increase the ability of the vector to transpose larger pieces ofDNA. Thus, we flanked an approximately 5 kb piece of DNA with two intactcopies of SB in an inverted orientation (FIG. 2A). The vector wasdesigned in a way that transposase was able to bind to its internalbinding sites within each element but its ability to cleave DNA at thosesites was abolished. Efficiency of transposition of the sandwich elementwas about 4-fold increased compared to an SB vector containing the samemarker gene (FIG. 2B) (unpublished results). Thus, the sandwichtransposon vector can be useful to extend the cloning capacity of SBelements for the transfer of large genes whose stable integration intogenomes has been problematic with current viral and nonviral vectors.

Sleeping Beauty integrates in a precise manner. Our analysis of ahandful, randomly chosen SB insertion sites in HeLa cells revealed thatchromosomal integration was precise in all of the cases, and wasaccompanied by duplication of TA target dinucleotides (Ivics et al.,1997), a molecular signature of Tc1/mariner transposition. To determinethe ratio of precise versus non-precise integration events in a laregerscale, a genetic assay for positive-negative selection was devised. Thisassay positively selects for integration of transposon sequences(precise events), and negatively selects against cells that carryintegrated vector sequences in their chromosomes (non-precise events).The thymidine kinase (TK) gene of herpes simplex virus type 1 was builtinto the vector backbone of pT/neo. Upon cotransfection of thisconstruct into cells together with a SB transposase-expressing plasmid,G-418-resistant colonies are selected either in the presence or absenceof gancyclovir, which is toxic to cells expressing the TK gene. About90% of the G-418-resistant Hela colonies survived gancyclovir selection,indicating that the majority of the integration events did not includethe toxic TK gene, which is a measure of precise, transposase-mediatedintegration events (FIG. 3). Similar results, indicative of precisetransposition, were obtained in hamster K1, fathead minnow FHM and mouse3T3 cells (Izsvak et al., 2000). Our results indicate a high fidelity ofsubstrate recognition and precise transposition of SB even in thesephylogenetically distant cell lines. The SB system provides preciseintegration of the desired gene, flanked by the short inverted repeatsequences (230 bp) only. This fidelity of integration means that plasmidsequences carrying antibiotic resistance genes are left behind and arenot integrating into the host genome, addressing a general problemconcerning gene therapy and transgenesis.

In contrast to concatamerization of extrachromosomal DNA, which is oftenencountered using nonviral gene transfer methods, SB transposonsintegrate as single copies.

SB can be expressed from a wide range of promoters to optimizetransposase expression for a variety of applications. Three differentpromoters were used to express SB transposase, those of the human heatshock 70 (HS) gene, the human cytomegalovirus (CMV) immediate early geneand the carp β-actin gene (FV). HS is inducible by applying heat shockon transfected cells, whereas CMV and FV can be considered as 'strong”constitutive promoter. As shown in the upper graph of FIG. 4, usingHS-SB and by increasing the time of the induction (15 min, 30 min and 45min), the numbers of G-418-resistant colonies increased as well. The CMVpromoter-driven transposase produced a significantly higher number ofcolonies, and we obtained even higher numbers with FV-SB (Izsvak et al.,2000). We assessed the relative strengths of the three promoters in geneexpression by measuring chloramphenicol acetyl transferase (CAT)reporter enzyme activity from transiently transfected cells. Levels ofCAT activity, when expressed from the same promoters under the sameexperimental conditions, showed about the same ratios as those weobtained for transpositional activities (FIG. 4, lower graph).

We concluded that the number of transposition events per transfectedcell population is directly proportional to the number of transposasemolecules present in cells. Thus, overexpression of transposase does notappear to have an inhibitory effect on SB transposition, at least not inthe range of expression in which SB would be used in most transgenicexperiments, and thus SB can be expressed from a wide range of promotersto optimize transposase expression for a variety of applications.

Sleeping Beauty transposon mediates the insertion of foreign genes intothe genomes of vertebrates in vivo. In contrast to viral vectors,tremendous quantities of plasmid-based vectors can be readily produced,purified and maintained at very little cost. Sleeping Beauty is is thefirst non-viral system that allows plasmid-encoded gene integration andlong-term expression in vivo.

Using naked DNA, tail-vein injection technique, Sleeping Beautytransposase was shown to efficiently mediate transposon integration intomultiple non-coding regions of the mouse genome in vivo. DNAtransposition occurs in approximately 5-6 percent of transfected mousecells and results in long term expression (>3 month) of therapeuticlevels of human clotting factor IX in vivo (Yant et al., 2000). Theseresults establish DNA-mediated transposition as a powerful new genetictool for vertebrates and provide intriguing new stategies to improveexisting non-viral and viral vectors for transgenesis and for human genetherapy applications.

The Sleeping Beauty inverted repeat sequences do not carry promoterand/or enhancer elements, which can potentially influence neighbouringgene expression upon integration into the genome. To test whether theinverted repeat sequence of the Sleeping Beauty transposon carriespromoter elements, the following experiment was performed. The lacZ genewas fused in frame to the SB transposase gene in a construct thatretained the transposon inverted repeat sequences upstream theexpression unit. Human HeLa cells transfected with this construct wereeither stained in situ or cell extracts were tested for β-galactosidaseactivity in an in vitro assay. No detectable β-galactosidase activitywas obtained in either case, suggesting that no significant promoteractivity could be rendered to the inverted repeats.

To test for enhancer activity, the left inverted repeat of the SBtransposon was fused to a minimal TK promoter in front of the luciferasemarker gene. The human cytomegalovirus (CMV) enhancer served as apositive control. No significant enhancer activity was observed from theinverted repeat sequence of Sleeping Beauty (unpublished results). Thus,in contrast to retroviruses whose LTRs contain enhancer/promoterelements, SB vectors are transcriptionally neutral, and thus would notalter patterns of endogenous gene expression.

Single amino acid replacements at nonessential positions in thetransposase polypeptide do not alter transposase activity. Eukaryoticexpression plasmids are all derivatives of the pCMV/SB constructdescribed earlier (Ivics et al., 1997). pCMV/SB-S116V was made byPCR-amplification of pCMV/SB with primers5′-CCGCGTCGCGAGGAAGAAGCCACTGCTCCAA-3′ and5′-CTTCCTCGCGACGCGGCCTTTCAGGTTATGTCG-3′,

cutting the PCR product with restriction enzyme NruI whose recogitionsequence is underlined within the primer sequences, andrecircularization with T4 DNA ligase. The mutant sequence with theencoded amino acids is the following: 109 110 111 112 113 114 115 116117 118 119 120 121 122 123 124 CGA CAT AAC CTG AAA GGC CGC GTC GCG AGGAAG AAG CCA CTG CTC CAA R H  N  L K  G  R V  A R K  K P  L L Q

The mutation is a single amino acid change in position 116, which is nowa valine (typed bold) in place of the original serine.

pCMV/SB-N280H was made by PCR-amplification of pCMV/SB with primers5′-GCCCAGATCTCAATCCTATAGAACATTTGTGGGCAGAACTG-3′ and5′-ATTGAGATCTGGGCTTTGTGATGGCCACTCC-3′,

cutting the PCR product with restriction enzyme BglII whose recogitionsequence is underlined within the primer sequences, andrecircularization with T4 DNA ligase. Part of he mutant sequence withthe encoded amino acids is the following: 270 271 272 273 274 275 276277 278 279 280 281 282 283 284 285 TCA CAA AGC CCA GAT CTC AAT CCT ATAGAA CAT TTG TGG GCA GAA CTG S Q  S  P D  L N P  I E  H  L W  A  E L

The mutation is a single amino acid change in position 280, which is nowa histidine (typed bold) in place of the original asparagine.

pCMV/SB-S58P was made by PCR amplification of a DNA fragment across thejunction of the CMV promoter and the transposase gene in pCMV/SB withprimers 5′-GGTGGTGCAAATCAAAGAACTGCTCC-3′ and5′-CAGAACGCGTCTCCTTCCTGGGCGGTATGACGGC-3′,

digestion with EagI which cuts at the junction of the CMV promoter andthe transposase gene and MluI (underlined), and cloning into therespective sites in pCMV/SB. Part of he mutant sequence with the encodedamino acids is the following:  54 55 56 57 58 59 60 61 62 G CCG TCA TACCGC CCA GGA AGG AGA CGC GT  P S  Y R  P  G R  R  R

The mutation is a single amino acid change in position 58, which is nowa proline (typed bold) in place of the original serine.

All constructs carrying the mutations were checked for proper expressionby Western hybridizations, using an anti-SB polyclonal antibody. Allthree above mutant transposases mediate transposition using the pT/neodonor construct (Ivics et al., 1997) in human Hela cells at comparablelevels to wild-type SB transposase (unpublished results). Comparablelevel is defined here being within a range of 90% to 110% of theactivity of wild-type SB transposase. Alltogether these data demonstratethat directed changes can be introduced into the transposase polypeptidewithout negatively affecting its functional properties. In summary, theSB system has several advantages for gene transfer in vertebrates:

-   -   SB can transform a wide range of vertebrate cells;    -   because SB is a DNA-based transposon, there is no need for        reverse transcription of the transgene, which introduces        mutations in retroviral vector stocks;    -   SB does not appear to be restricted in its ability to transpose        DNA of any sequence;    -   SB vectors do not have strict size limitations;    -   since transposons are not infectious, transposon-based vectors        are not replication-competent, herefore do not spread to other        cell populations;    -   SB requires only about 230 bp transposon inverted repeat DNA        flanking a transgene on each side for mobilization;    -   SB vectors are transcriptionally neutral, and thus do not alter        endogenous gene function;    -   transposition is inducible, and requires only the transposase        protein, thus one can simply control the site and moment of        jumping by control of transposase expression.    -   SB is expected to be able to transduce nondividing cells,        because the transposase contains a nuclear localization signal,        through which transposon/transposase complexes could be actively        transported into cell nuclei;    -   SB mediates stable, single-copy integration of genes into        chromosomes which forms the basis of long-term expression        throughout multiple generations of transgenic cells and        organisms;    -   once integrated, SB elements are expected to behave as stable,        dominant genetic determinants in the genomes of transformed        cells, because 1) the presence of SB transposase is only        transitory in cells and is limited to a time window when        transposition is catalyzed, and 2) there is no evidence of an        endogenous transposase source in vertebrate cells that could        activate and mobilize integrated SB elements;    -   with the exception of some fish species, there are no endogenous        sequences in vertebrate genomes with sufficient homology to SB        that would allow recombination and release of transpositionally        competent (autonomous) elements;    -   for efficient introduction into cells, SB could be combined with        DNA delivery agents such as adenoviruses and liposomes;

because SB is a plasmid-based vector, its production is easy,inexpensive, and can be scaled up. TABLE 1 Transposase Class OrganismCell line − + Activity Mammals Human Hela 282 8750 +++++ Jurkat 2 6 +Monkey Cos-7 885 1845 + Mouse LMTK 155 805 ++ 3T3 170 850 ++ ES(AB1)^(#) ++ Hamster K1 8250 87900 ++++ Rabbit SIRC 174 318 + DogMDCK-II 22 50 + Cow MDBK 480 4185 +++ Sheep MDOK 13 27 + Birds Quail QT64 3 ? Amphibians Xenopus A6 12 252 +++++ Fishes Zebrafish ZF4 7 13 +Carp EPC 54 129 + Sea bream SAF1 9 13 + Medaka OLF136 10 34 ++ Trout RTG4 13 + Swordtail A2 37 108 + Fathead minnow FHM 4 104 +++++

1. A method for using the Sleeping Beauty gene transfer system forsomatic gene transfer for stable insertion of DNA into chromosomes ofliving vertebrates comprising: the two components of the Sleeping Beautytransfer system which are transferred into the somatic cells of ananimal for gene therapeutic purposes.
 2. Wherein the method of claim 1is used for diagnostic purposes.
 3. The method of claim 1 wherein thetransferable gene inserted into the Sleeping Beauty transposon is fromhuman.
 4. The method of claim 1 wherein the transferable gene insertedinto the Sleeping Beauty transposon is from fish.
 5. The method of claim1 wherein the transferable gene inserted into the Sleeping Beautytransposon is from frog.
 6. The method of claim 1 wherein thetransferable gene inserted into the Sleeping Beauty transposon is fromreptile.
 7. The method of claim 1 wherein the transferable gene insertedinto the Sleeping Beauty transposon is from bird.
 8. The method of claim1 wherein the transferable gene inserted into the Sleeping Beautytransposon is from mammals.
 9. The method of claim 1 wherein thevertebrate is a human.
 10. The method of claim 1 wherein the vertebrateis a fish.
 11. The method of claim 1 wherein the vertebrate is a frog.12. The method of claim 1 wherein the vertebrate is a reptile.
 13. Themethod of claim 1 wherein the vertebrate is a bird.
 14. The method ofclaim 1 wherein the vertebrate is a mammal.
 15. The method of claim 1wherein the transposase source is a transposase protein.
 16. The methodof claim 1 wherein the transposase source is an mRNA.
 17. The method ofclaim 1 wherein the transposase source is a plasmid DNA.
 18. The methodof claim 1 wherein the delivery method is a gene gun.
 19. The method ofclaim 1 wherein the Sleeping Beauty transfer system is combined withliposomes.
 20. The method of claim 1 wherein the Sleeping Beautytransfer system is combined with PEI (polyethylene Imine).
 21. Themethod of claim 1 wherein the Sleeping Beauty transfer system iscombined with adenovirus-polylysine-DNA complexes-receptor-mediated genetransfer.
 22. The method of claim 1 wherein the Sleeping Beauty transfersystem is combined with a recombinant retrovirus.
 23. The method ofclaim 1 wherein the delivery method is transduction.
 24. The method ofclaim 1 wherein the Sleeping Beauty transfer system is combined with arecombinant adenovirus.
 25. The method of claim 1 wherein the deliverymethod is infection.
 26. The method of claim 1 wherein the SleepingBeauty transfer system is combined with a recombining herpes virus. 27.The method of claim 1 wherein the Sleeping Beauty transfer system iscombined with a recombinant adeno-associated virus.
 28. The method ofclaim 1 characterised in that the transferable gene is flanked by twocomplete SB elements in either direct or inverted orientation withrespect to each other, characterised in that an SB element is defined bytwo IR/DR sequences in an inverted orientation with respect to eachother, each containing at least two transposase binding sites.
 29. Themethod of claim 1 characterised in that the transposase has at least a90%, preferably a 95%, more preferably a 98% sequence identity to SEQ1.30. The method or claim 1 wherein the transferable gene is used forcorrecting a single gene defect.
 31. The method of claim 1 wherein thetherapeutic gene is used in cancer gene therapy.
 32. The method forusing the Sleeping Beauty transfer system for germ line gene transfer invertebrate organisms comprising: a. the two components of the SleepingBeauty transfer system which are introduced into the germ stem cell toyield a transgenic cell. b. growing the transgenic cell into atransgenic animal.
 33. The method of claim 32 wherein the cell isplurlpotent or totipotent.
 34. The method of claim 32 wherein deliverymethod is microinjection.
 35. The method of claim 32 wherein thedelivery method is gene gun.
 36. The method of claim 32 wherein thedelivery method is germ transport of the Sleeping Beauty transfersystem.
 37. The method of claim 32 characterised in that thetransferable gene is flanked by two complete SB elements in eitherdirect or inverted orientation with respect to each other, characterisedin that an SB element; is defined by two IR/DR sequences in an invertedorientation with respect to each other, each containing at least twotransposase binding sites.
 38. The method of claim 32 characterised inthat the transposase has at least a 90%, preferably a 95%, morepreferably a 98% sequence identity to SEQ1.
 39. The method of claim 32wherein the transferable gene inserted into the Sleeping Beautytransposon is from human.
 40. The method of claim 32 wherein thetransferable gene inserted into the Sleeping Beauty transposon is fromfish.
 41. The method of claim 32 wherein the transferable gene insertedinto the Sleeping Beauty transposon is from frog.
 42. The method ofclaim 32 wherein the transferable gene inserted into the Sleeping Beautytransposon is from reptile.
 43. The method of claim 32 wherein thetransferable gene inserted into the Sleeping Beauty transposon is frombird.
 44. The method of claim 32 wherein the transferable gene insertedinto the Sleeping Beauty transposon is from mammals.
 45. The method ofclaim 32 wherein the vertebrate is a human.
 46. The method of claim 32wherein the vertebrate is a fish.
 47. The method of claim 32 wherein thevertebrate is a frog.
 48. The method of claim 32 wherein the vertebrateis a reptile.
 49. The method of claim 32 wherein the vertebrate is abird.
 50. The method of claim 32 wherein the vertebrate is a mammal. 51.The method of claim 32 wherein the transposase source is a transposaseprotein.
 52. The method of claim 32 wherein the transposase source is anmRNA.
 53. The method of claim 32 wherein the transposase source is aplasmid DNA.